Method and device for refrigerating a sample

ABSTRACT

To improve the thermal transmission during freezing processes it is proposed that the goods to be cooled and a pre-cooled body of high thermal capacity be pressed against each other. 
     Improved thermal transmission is also attained with a device comprising a holding device which during the cooling process holds the goods to be cooled essentially in a non-deformable way, allowing direct contact between the coolant and the goods to be cooled. Preferably, vertically aligned channels are arranged between the holding device and the goods to be cooled, with coolant flowing in said channels.

The invention relates to a device for cooling, in particular freezing, goods to be cooled, in particular biological materials.

Devices for freezing biological materials are known from cryobiology.

In many fields of biological specimen preparation as well as intense-cooling conservation or vitrification of cells, organs or organisms or other biological materials, for various reasons it is crucial to cool specimens as fast as possible.

In the case of biological specimen preparation by cryotechniques e.g. for histology examinations, it is important that the specimen morphology be maintained as far as possible in spite of cooling the specimen. This requires rapid cooling to keep the extent of ice formation to a minimum.

Devices for freezing blood cells are particularly well researched. With these devices, antifreeze additives such as for example hydroxyethyl starch (HES) or glycerine is added to the blood components; said additives are necessary to achieve an adequate cell survival rate after the freeze-thaw process. The blood components present in a film bag are placed in a container which is subsequently cooled by immersion in e.g. liquid nitrogen.

To achieve a good ratio of surface to volume at the bag and to avoid creases and bulges at the bag, DE 31 42 521 C2 and DE-A-44 37 091 propose that the bag be held between two plates arranged parallel in respect of each other, and that the bag be cooled in the liquid nitrogen together with the holding device. A similar holding device is also known from WO 90/09184.

From U.S. Pat. No. 4,018,911 a holding device is known where the bags are positioned loosely in a perforated holding device so that the coolant can reach the bags also through the holding device.

However, the known holding devices have a disadvantage in that thermal transmission from the coolant to the biological material is impeded by the holding device.

It is thus the object of the invention to provide a device of the generic type which allows acceleration of the cooling process of goods to be cooled.

The object of the invention is met by a generic device comprising a holding device which during the cooling process holds the goods to be cooled essentially in a non-deformable way and forms at least one space which allows direct contact between the coolant and the goods to be cooled, with channels for conducting the coolant being provided between the holding device and the goods to be cooled.

Preferably, the space extends at least partly along the goods to be cooled.

In the case of boiling coolants, the guide channels for conducting the coolant allow free convection or pumping of the coolant through the channels.

The holding device according to the invention is constructed such that it holds in particular liquid blood components filled into bags in a shape that is advantageous for cooling while nevertheless allowing direct contact between the coolant and the goods to be cooled. This not only results in optimum holding of the goods to be cooled but also in avoiding the problems, known from the state of the art, of thermal transmission from the goods to be cooled through the holding device into the coolant.

To achieve good thermal transmission from the goods to be cooled to the coolant, it is proposed that the contact surface between the holding device and the goods to be cooled be smaller than the contact surface between the coolant and the goods to be cooled.

Trials have shown that convection through evaporation itself, within the described guide channels, results in strong acceleration of the coolant if the inlet of the guide channels is arranged lower than their outlet.

This chimney effect is so pronounced that it is proposed that flow-control devices which are adjustable so as to restrict the flow, be arranged at the inlet and/or the outlet. This makes it possible to achieve control or regulation of the coolant flow in a simple way. It is advantageous if the adjustable flow-control devices are adjustable also during the cooling process, so that variations in the cooling rates between the margin and the middle of the specimen can be compensated for by regulation, because otherwise different local survival rates could result.

Simple construction of the device is achieved in that the holding device holds the goods to be cooled in the form of a plate. In particular good cooling rates can be achieved by cooling both sides of the plate-shaped goods to be cooled.

The goods to be cooled can however also be held in the form of a cylinder. Particularly advantageous is a holding device which holds the goods to be cooled in the form of a hollow cylinder because in this way the goods to be cooled encompass a hollow space which can serve as a guide channel for the coolant.

Since in the range of the boiling temperature of the coolant the best cooling rate can be achieved with nucleate boiling, a chamber is proposed into which the holding device can be inserted, with said chamber preferably being heatable. The ability to heat the chamber allows precise setting of the evaporation rate of the coolant and thus of the convection. Alternatively or additionally, the holding device itself can be constructed so as to be heatable.

It is advantageous if the chamber comprises an inlet with a coolant pump. In this way forced flow of the coolant through the chamber and between the goods to be cooled and the holding device can be achieved, with said forced flow improving the thermal transmission from the goods to be cooled to the coolant.

A preferred embodiment provides for the chamber to comprise an overflow and a separator for liquid coolant. While the liquid coolant is used for further cooling, the gaseous part of the coolant is either discarded or liquefied in a connected device.

A preferred use of the device described comprises the freezing of bags filled with a liquid, in particular blood components. These bags are flexible in shape; they have to be cooled as fast as possible. Although antifreeze additives limit the damage to blood components, particularly high rates of cooling should be achieved. This can be achieved in a simple way with the device described.

When cooling bodies and in particular when cooling liquids, the volume of the goods to be cooled changes, in the case of aqueous systems in addition also due to crystallisation.

It is thus proposed that the holding device described essentially be pressed against the goods to be cooled, at a constant pressure. This can for example be achieved by a pre-tensioned spring with flat spring characteristics, with pneumatic or hydraulic devices. In particular, a hydraulic or pneumatic device with respective control makes it possible to keep the pressure against the goods to be cooled essentially constant. Although the volume increase during crystallisation can principally be absorbed by regulating the pressure, it is additionally advantageous if the film bag is not completely filled, but instead, if a gas cushion is left above the goods to be frozen. In this way the welded seams of the bag are not unduly stressed by the expansion in volume.

In order to improve the thermal transmission at the holding device, it is proposed that the holding device comprise a microporous surface on the side of the coolant. To form a microporous surface, either the surface itself can be roughened or an adhesive layer with a microporous surface, for example Leukosilk® can be applied to the surface. It is particularly advantageous if this microporous layer is fixed directly to the bag.

Depending on the device selected, or depending on the desired cooling progression, prior to cooling the goods to be cooled, the temperature of the holding device can be below the solidification temperature of the goods to be cooled. But it is also possible that the temperature of the holding device is above the solidification temperature of the goods to be cooled.

To illustrate the device described, several embodiments are shown in the drawing and are described in more detail below. The following are shown:

FIG. 1 a diagrammatic lateral view of a device with a holding device with cooling ribs;

FIG. 2 a section through the device according to FIG. 1;

FIG. 3 a diagrammatic view of a device for free convection with heating device; and

FIG. 4 an alternative embodiment of a device for free convection with heating device.

FIG. 5 a section through the device according to FIG. 4;

FIG. 6 a diagrammatic view of a device for free convection with heating device; and

FIG. 7 an alternative embodiment of a device for free convection with heating device.

FIG. 1 shows a further device 11 for freezing or cooling a cooling bag 12. The bag is jammed between two L-shaped plates 13 and 14 which completely encompass the bag 12. The plates 13 and 14 are pressed together by way of pneumatic cylinders 15, 16, such that the cooling bag 12 is firmly held between the plates.

On their sides facing the bag 12, the plates 13 and 14 comprise a comb-like structure, shown in FIG. 2. In this way channels 17 form when the plates 13, 14 are pressed against the cooling bag 12, with coolant being able to rise in said channels 17, along the arrows 18, between the plates and the cooling bag 12.

The plates 13, 14 and the cooling bag 12 are arranged in a chamber 19 which is closed by a lid 20. The plates are arranged at a distance from the bottom of the chamber so as to allow streaming movement below the plate. By way of the pipe 21 and the pump 22, liquid nitrogen is pumped into this chamber. This liquid nitrogen first accumulates on the bottom of the chamber 19 before rising in the channels 17. In so doing it heats up and changes to the vapour phase. The channels 17 have a chimney effect leading to a particularly strong flow within the channels. In order to regulate this flow, flaps 23, 24 are provided at the entrance of the channels 17. The nitrogen emanating from the upper end of the channels 17 flows to a separator 25 which separates liquid nitrogen from gaseous nitrogen. The separator 25 comprises a vapour exit aperture through which the gaseous nitrogen is educted.

In those positions where the plates 13 and 14 are arranged directly between the coolant and the cooling bag 12, a microporous layer 26, 27 is provided which improves thermal transmission from the coolant to the plate and thus thermal transition to the cooling bag. The cooling performance could be further improved if the surface of the cooling bag 12, at least in the region of the channels 17 and/or further surface regions of the plates 13, 14 which are in contact with the liquid nitrogen, comprises a microporous layer.

FIG. 3 is a diagrammatic arrangement of a device according to FIG. 1 with heating elements 28 and 29 which are arranged on the sides of the plates 31 and 32, said sides facing the cooling bag 30. In this way the flow speed in the channels 34, 35 can be increased and regulated. A further heating element 36 is provided in the bottom region of the device, again so as to improve and regulate the flow of the coolant. In this variant, the liquid part of the coolant emerging upward is collected in a device (not shown).

FIG. 4 shows a further alternative embodiment of the device according to FIG. 1 comprising two heating devices 37, 38 for the holding devices 43, 44. In this variant, the coolant moves downward in the exterior region of a container 39; it is conducted to the channels 41 and 42 in the plates 43 and 44 by way of a funnel 40. Above the channels 41 and 42, deflectors 45, 46 are provided which return liquid coolants issuing from the channels 41, 42. Above the deflectors 45, 46, a lid 47 with a gas outlet 48 is provided. The liquid level 49 of the coolant is kept just above the cooling bag but below the deflectors 45, 46. In this way the consumption of liquid nitrogen is reduced.

In the embodiments according to FIG. 3 and 4, too, the lower inlet of the channels can be regulated by flaps.

The embodiments according to FIGS. 1, 2, 3 and 4 can either be operated so that the goods to be cooled and the container or the plates are at first held above the solidification temperature of the goods to be cooled, or the container or the plates can already be precooled and only the goods to be cooled can be held above the solidification temperature before they are placed into the device. 

What is claimed is:
 1. A sample-cooling device comprising at least one precooled body delimiting a cooling space for the sample, wherein at least one precooled body has a mass in grams that is at least 2.5 times the volume of the cooling space in milliliters, and wherein the precooled body comprises at least one space which permits formation of a contact surface and allows direct contact between a coolant and the sample.
 2. A device for cooling a sample, the device comprising: (a) a holding device arranged: (i) to hold the sample in an essentially non-deformable manner; and (ii) to form at least one contact surface between the holding device and the sample; and (b) at least one space which permits formation of a contact surface and allows direct contact between a coolant and the sample.
 3. The device of claim 2 wherein the contact surface between the holding device and the sample is smaller than the contact surface between the coolant and the sample.
 4. The device of claim 2 wherein the holding device comprises guide channels for conducting the coolant.
 5. The device of claim 4 wherein the guide channels comprise an inlet and an outlet and wherein the inlet is positioned at a level which is lower than the level of the outlet.
 6. The device of claim 5 further comprising at least one adjustable flow-control device arranged to restrict flow of coolant at the inlet and/or the outlet.
 7. The device of claim 2 wherein the holding device is arranged to hold a plate-shaped sample.
 8. The device of claim 2 wherein the holding device is arranged to hold a sample having a cylindrical or a hollow-cylindrical shape.
 9. A device for cooling a sample, the device comprising: (a) a holding device arranged: (i) to hold the sample in an essentially non-deformable manner; and (ii) to form at least one contact surface between the holding device and the sample; (b) at least one space which permits formation of a contact surface and allows direct contact between a coolant and the sample, and (c) a heatable chamber surrounding the holding device.
 10. The device of claim 9 wherein the chamber comprises an inlet for flowing coolant into the chamber.
 11. The device of claim 9 wherein the chamber comprises an overflow and a separator for liquid coolant.
 12. The device of claim 2, wherein the sample is a freezing bag filled with a liquid.
 13. The device of claim 12, wherein the freezing bag comprises a microporous surface.
 14. The device of claim 12, wherein the holding device is kept at a temperature that is below the solidification temperature of the liquid in the freezing bag prior to cooling of said freezing bag.
 15. The device of claim 12, wherein the holding device is kept at a temperature that is above the solidification temperature of the liquid in the freezing bag prior to cooling of said freezing bag.
 16. The device of claim 2, wherein the holding device is pressed against the sample at an essentially constant pressure.
 17. The device of claim 2, wherein the contact surface between the holding device and the sample comprises a microporous surface. 